We have reported a high prevalence of hypothyroidism within the melanoma population and hypothesize that thyroid stimulating hormone (TSH), which circulates at high concentrations in hypothyroid individuals, promotes melanoma growth. This concept is supported by preliminary data showing that melanoma cells express functional TSH receptors (TSHR) and that TSH induces proliferation of cultured melanoma cells. The research described in this proposal will test the hypothesis that signaling of TSH through its receptor on human melanoma cells promotes tumor growth and progression; and that this mitogenic process is enhanced by elevated circulating levels of TSH and over-expression of melanoma TSHR. There are three Specific Aims. Specific Aim 1 uses saturation radioligand binding experiments with cultured melanoma cells to examine the density and affinity of melanoma TSHR, and compare the findings to thyrocytes. If a high density of melanoma TSHR is found, the cells will be examined for amplification of the TSHR gene by fluorescent in situ hybridization (FISH). Experiments will also be conducted determine if melanoma TSHR activate three pathways typical of thyrocyte TSHR signaling. These pathways, which include the protein kinase A (PKA), mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K) cascades, will be examined by western blotting for phosphoproteins and signal protein translocation in response to TSH. Specific Aim 2 encompasses a clinical study of 150 high risk Stage II melanoma patients who have had their primary tumors excised. These patients have a 30% rate of relapse and routinely undergo sentinel node biopsy (SNB) to detect early lymphatic metastases. TSH levels will be measured in the study patients prior to SNB, with the prediction that patients with tumor-positive sentinel nodes will have higher TSH levels than those with negative nodes. In Specific Aim 3, paraffin embedded primary tumors from the Aim 2 study cohort will be used to prepare a tissue microarray. Sections of the array will be examined by immunostaining for TSHR density, and findings will be correlated with disease progression. Similar to Aim 1, if a high density of TSHR are found in the melanoma tissues, FISH will be used to determine the presence of TSHR gene amplification. If our hypothesis is proven to be correct, this research will open a new and completely unexplored area involving the promotion of melanoma growth by TSH, with wide implications for screening of melanoma patients for hypothyroidism, and for aggressive management of suspicious cutaneous lesions in hypothyroid individuals. Moreover, melanoma TSHR may ultimately prove to be a direct therapeutic target, or be exploited as a means of delivering antibody- or hormone-complexed drugs. [unreadable] [unreadable] This project examines the biologic mechanisms to account for the association of melanoma with hypothyroidism. Medications used to treat hypothyroidism may actually be therapeutic for melanoma. Furthermore, melanoma cases may be prevented by heightened surveillance of individuals with a history of hypothyroidism. [unreadable] [unreadable] [unreadable]